The discovery of the polymerase chain reaction (PCR) has revolutionised the field of molecular biology allowing for the amplification of any desired stretch of DNA from any organism.
More specialised use of PCR is seen in the areas of real-time quantitation and in end point determination of Genotypes where the use of homogeneous assay systems is employed. A homogeneous assay system is one where to derive the result of the reaction does not require the physical separation of the reaction components away from each other. In other words the reaction is conducted and the results derived with no further physical intervention into the reaction.
When monitored in real-time (in other words, monitoring the production of product at every cycle during the PCR process) PCR can be used in a quantitative manner. This has wide application in many fields from diagnosis of viral infections to determination of the abundance of a messenger RNA species within human RNA. The physical process of monitoring real time PCR is complex, requiring sophisticated instrumentation specifically designed for the process. This instrumentation requires the PCR process to produce a measurable amount of light increasing with every cycle until the reaction components are exhausted. To serve this need a number of elegant approaches have been demonstrated and commercially exploited. Two such examples of these are the Taqman 5′ nuclease assay marketed by Applied Biosystems (USA) and the SybRGreen detection chemistry marketed by Molecular Probes (Netherlands). Both Quantitative PCR systems work well, but both have drawbacks that would benefit from being overcome.
Genotyping (in particular Single Nucleotide Polymorphism (SNP) Genotyping) on the other hand is a process that is less demanding on the instrumentation. The process is essentially the same employing the use of a homogeneous assay system, but the requirement for monitoring the reaction products at each cycle of the PCR is less critical. Indeed, the majority of scientists conducting SNP Genotyping using fluorescence based systems conduct the PCR and only at the end of the reaction do they determine the levels of product made. This is generally termed end-point analysis. SNP Genotyping has a further level of complexity, in that the purpose of the reaction is to determine the individual DNA genotype at a single locus within their genome. SNP's are biallelic markers that are ideally suited to being determined by fluorescent homogeneous assays in large numbers at low cost. The various reaction systems that are currently in use are again the Taqman reaction (Applied Biosystems), the Amplifluor system (Serologicals, USA) and the Scorpions system (DxS, UK). All are elegant reaction systems producing good quality data, but each has its own drawback.
The principle of all the homogeneous assay systems is to use the physical process of Fluorescence Resonance Energy Transfer (FRET) to detect the production of product in the PCR process. FRET is the process whereby when two fluorophores are in close enough proximity to each other that they will undergo an energy transfer exchange when excited by light at wavelengths matched to their particular excitation wavelength. FRET is ideally suited to the quantitation of PCR as it allows for the reaction to be monitored without being separated, a process that would be impossible bearing in mind that in general 40 cycles of PCR are carried out and the amount of product needs to be determined after each cycle.
The main techniques currently in use work well but suffer a number of drawbacks that hamper their use in the scientific world.
The Applied Biosystems Taqman assay as discussed in, for example, U.S. Pat. No. 5,538,848 (or, more generally, a FRET based 5′ nuclease assay), requires the production of a dual labelled Oligonucleotide probe for each DNA sequence to be measured. This probe in its pre-reaction state is termed ‘quenched’. In other words two fluorophores (or one non-fluorescent quencher and a fluorophore) are attached to a short Oligonucleotide within approx 30 nucleotides of each other. This distance is small enough that when one of the fluorophores is excited at its optimal wavelength the other fluorophore absorbs the energy absorbed and emits light at a different wavelength, or in the case of a non-fluorescent quencher the absorbed energy is passed by FRET to the non-fluorescent quencher and no light is emitted. When this molecule is included in the reaction the PCR process creates DNA complementary to it. This allows the probe to bind to the DNA whereby it is subsequently destroyed by the 5′ nuclease activity of the Taq polymerase used in the PCR process. Now the probe is degraded the two fluorophore pairs are no longer in close enough proximity to undergo FRET and a measurable light difference is created.
The main drawback with the Taqman assay is the requirement for the production of the dual labelled Oligonucleotide probes. A single probe is required for quantitative measurements of DNA mass, whereas two are required for SNP genotyping (one for each allele). The production of the probe itself is a costly and timely process. At time of writing each probe can cost as much as £250, yielding enough reagent for only a few thousand reactions. If one is to consider that in a SNP Genotyping project anywhere upwards of 200 SNP's can be studied, then this would require an initial investment of £10,000 in probe production. This is a prohibitively large sum for many science organisations, and thus a major drawback of the system.
In the area of quantitative gene expression the use of SYBR Green is often employed as a low cost alternative to the use of Taqman. SYBR Green is an interchelating dye that only binds double stranded DNA. As such it can be employed in quantitative homogeneous PCR assays. The PCR product is generated as the cycle number in the reaction increases, and as the product builds up the SYBR Green binds to the product. Once bound the SYBR Green undergoes a conformational change and exhibits fluorescence which is directly measured. The main two drawbacks to the use of this technique are the non-specific nature of the reaction, in that any product whether it is the correct product or not will produce a signal. It is therefore imperative to confirm that the PCR produces the amplicon that is required to be measured. Taqman does not suffer from this drawback as the probe interacts with the sequence of the correctly generated amplicon only. Secondly, the use of SYBRgreen is known to be difficult to optimise as the SYBR green itself can interact with the PCR process making the reaction difficult to optimise.
The Amplifluor and Scorpion homogeneous PCR assay systems also suffer from similar drawbacks. Both systems utilise a tailed PCR primer to interact with a hairpin quenched fluorescent primer. In other words the PCR reaction is initiated with conventional oligonucleotide primers of which one (or two in the case of allele specific PCR based SNP genotyping) contains a sequence that is identical to the 3′ end of the Amplifluor or Scorpion fluorescent primers. The reverse complement to this sequence is then made during the first few cycles of the PCR reaction. This allows the fluorescent primers to then initiate the PCR reaction. Upon doing this the hairpin structure of the Amplifluor or Scorpion primers is copied and ‘unravelled’. These hairpin structures contain fluorescent quencher pairs, that are fully quenched when the hairpin structure can form, however when this is copied the structure can no longer form, and hence the fluorophore quencher pair is separated and fluorescent signal is generated. A number of modes of operation exist for both Amplifluor and Scorpion technology, however they suffer from at least one or more of the following drawbacks. Each hairpin-based primer is again difficult and costly to synthesise, due to the complex nature of the hairpin and dual labelled fluorophore quencher pairs. This cost and technically demanding nature of the synthesis is a major drawback. Further to this the reaction is also susceptible to the generation of signal from non-specific PCR artefacts such as primer dimer, and incorrect amplicon generation.
Further alternative assays are disclosed in the following patent applications: PCT WO00/41549; EP 0,909,823; PCT WO02/30946; PCT WO99/49293; and DE 10230948.
The need for an easy to synthesise, low cost and relatively reliable specific detection system for homogeneous PCR assays is apparent. It is a general objective of the present invention to address one or more of these aforementioned shortcomings of the existing FRET-based detection systems for PCR. The following invention addresses these in a number of different formats, providing a detection system suitable for the detection of PCR products directly or indirectly and which may be used in a quantitative, real-time and/or end point manner.